In the mycotic diseases, there are superficial one which develops on the skin and deep-seated one which develops in the internal organ, blood system and lymph system. The deep-seated mycotic disease is a kind of opportunistic infectious disease with which patients who lost their power of resistance (immunodeficiency state) are affected, and leads to extremely serious pathological condition. A representative example of causative microorganism of the deep-seated mycotic disease includes species of Candida and Aspergillus, and since the βG is present commonly in the cell walls of both species, it is useful to determine blood level of the βG. In the clinical diagnosis, determination of plasma or serum β-glucan level is used for earlier diagnosis of fungal infection, determination of therapeutic effect and prognosis.
The βG has a structure of repeated glucose unit linked by β-(1→3) bond as a main chain, and in some cases it has branched structure with (1→6) bond or (1→4) bond, and has high molecular weight of about several thousands to millions (in this regard, however, distribution is wide). In addition, the βG has a property of binding with βG-binding domain of factor G-subunit α which is present in the hemocyte extract (amebocyte lysate) of horseshoe crab.
The structural analysis of the Tachypleus horseshoe crab factor G-subunit α is completed, and its amino acid sequence and nucleotide sequence have been disclosed in the NCBI (National Center for Biotechnology Information) database. In addition, expression of Tachypleus horseshoe crab factor G-subunit α by genetic engineering technique has also been succeeded (Patent Literature 3).
The factor G is a precursor of serine proteases, and is activated by binding with βG to initiate a protease cascade of factor G series. And the activated factor G activates proclotting enzyme to clotting enzyme, and finally this results in gel formation. Consequently, in the field of medical, pharmaceutical and microbiological science, the βG detection methods have been developed by utilizing this property of the hemocyte extract of horseshoe crab.
As for the representative example of βG detection method employed primarily at the moment, gelation test and turbidimetric kinetic method through the use of gelation reaction arising out of a solution containing hemocyte extract of horseshoe crab and βG, as well as endpoint synthetic substrate method, kinetic synthetic substrate method and the like are included.
For example, the determination of βG by the turbidimetric kinetic method is carried out as follows. That is, a reagent containing hemocyte extract of horseshoe crab is mixed with a sample containing βG, and the aforementioned mixture is irradiated by light. Subsequently, using an appropriate measuring instrument (for example, spectrophotometer, microplate reader, etc.), the time length of optical change, such as change in transmittance, change in absorbance, variation in a ratio of transmitted light Rt, variation in logarithmic value of the ratio of transmitted light Rt, and the like, required for arriving at a predetermined value (gelation time, Tg) after initiation of light irradiation for the aforementioned mixture is measured. The βG concentration in the sample is determined by fitting the aforementioned time obtained to a standard curve indicating relationship between gelation time and βG concentration which has been made in advance using βG solutions of known concentrations.
The measurement methods using hemocyte extract of horseshoe crab as mentioned above are a method which enables to perform measurement of small quantity of βG in the blood in high sensitivity. However, these methods, on the other hand, have such problems that (1) the measurement results tend to vary depending on tester because the method is carried out manually; (2) due to specific measurement for the βG in the blood, it is necessary to carry out inactivation of endotoxins in the blood or to make the test reagents endotoxin-insensitive; (3) in order to inactivate interfering factors in the blood which interfere the horseshoe crab cascade reaction, it is necessary to carry out pre-treatment of the test sample; (4) the measurement takes a long measurement time (about 90 minutes); (5) since hemocyte extract of horseshoe crab is used, that is, natural material (naturally occurring substance) is used, there is concern about depletion of resources; (6) since hemocyte extract of horseshoe crab is used, that is, natural material is used, it costs to maintain quality of the material stable, and so on.
And so, to solve the above described problems in the measurement methods using protease cascade, some additional new measurement methods have been developed.
For example, there is a method in which a protein consisting of only βG binding domain of the factor G-subunit α is prepared by genetic engineering technology and fluorescently labeled, and using one molecule of the fluorescently-labeled recombinant protein instead of using hemocyte extract of horseshoe crab which is natural material, βG is measured by fluorescence polarization technique (Patent Literature 1). However, the detection sensitivity of this method is about several ng/mL at the highest of the reduced value of pachyman (a kind of βG), and this is insufficient to apply the method to the clinical laboratory tests.
In addition, a method of measurement based on Biacore technique using sensor chip (Non-Patent Literature 1), a method for measuring βG using βG-binding protein having a property of specifically binding to βG and a property of inhibiting activation of horseshoe crab factor G, and an antibody against βG-binding protein which is labeled with a labeling substance (Patent Literature 2) have been reported. It is described in the Patent Literature 2 that the βG-binding protein can be produced by genetic engineering technology.
Although the method using sensor chip measures principally affinity between βG and βG-binding protein, since the measurement is qualitative and operation is cumbersome, there remains a problem in applying this method to the field of laboratory testing.
In addition, since the βG-binding protein used in the method which uses βG-binding protein having a property of inhibiting activation of horseshoe crab factor G and an antibody against βG-binding protein which is labeled with a labeling substance is a natural product, there is a problem that lot difference may occur. In addition, it has not been studied yet whether this method can be applied to the field of laboratory testing. Further, this method has a problem in sensitivity.
Still further, an invention relating to “A kit for detecting fungi, comprising one domain or a plurality of domains derived from a Z1 domain and/or a Z2 domain of a factor G subunit α Xln for horseshoe crab of which the cysteine residue is replaced by other amino acid, and comprising a recombinant protein capable of binding to β-1,3-glucan” has also been known (Patent Literature 4).
This method is a method for detecting fungi by contacting a kind of recombinant protein having the above described property with a sample comprising fungi. However, a method for measuring βG using the aforementioned protein (including sandwich method) has not been studied.
Using two molecules of Dectin 1 which is known as a βG receptor, a method for measuring βG by ELISA and the like is also known (Non-Patent Literature 2). However, this method has also not attained to the level of sensitivity required for laboratory testing.
Further, among bacteria such as cellulolytic bacterium of Fibrobacter succinogenes, one which possesses cellulose-binding protein is known. A region in the cellulose-binding protein where the protein binds to cellulose is referred to as cellulose binding domain (CBD). The CBD is further classified into plural subfamilies (CBD I to X) according to the characteristic of an amino acid sequence (Advances in Microbial Physiology, vol. 37, p. 1-81, 1995). In CBDs, there are ones which have a xylanase Z-like domain having property of binding to βG. However, not necessarily all CBDs have the xylanase Z-like domain. In addition, cellulose which is a target of binding by CBD is (1→4)-β-D-glucan, however, βG in plasma which is a measurement object in clinical testing is (1→3)-β-D-glucan. Therefore, it has not been clarified whether the CBD can be utilized for measuring βG in the field of clinical testing even if the one which have the xylanase Z-like domain is present in the CBD.
As described above, all these methods had various problems when βG was intended to be measured specifically, and particularly, to utilize the method for clinical testing.